This invention relates to the field of immobilizing proteins upon polymeric supports, particularly to the immobilization of enzymes upon porous polymeric materials.
Enzymes, the proteinaceous catalysts for many biological reactions, have wide use in the food and pharmaceutical industries. For example, glucose isomerase is used for the conversion of a glucose reaction mixture to fructose, lactase for the removal of lactose during the isolation of proteins from cheese whey, .alpha.-amylase and glucoamylase for the liquefaction and saccharification of corn starch to liquid syrup, and penicillin amidase to produce 6-aminopenicillanic acid from penicillin. The immobilization of enzymes on solid supports has advantages that have long been recognized. Upon treatment with the catalyst by passage of a mixture or substrate through the support, the immobilized enzyme remains bonded to the support rather than passing through with the substrate so that there is no need to recover the enzyme from the substrate and so that the enzyme remains in the support where it may be reused.
Prior art methods of immobilizing proteins on solid supports include that disclosed in U.S. Pat. No. 4,210,722, issued to Silver on July 1, 1980, and entitled "Protein Immobilizer", hereinafter referred to as Silver. Silver discloses a method of immobilizing a biologically active protein on a polar support by first applying to the support a monolayer or coating of a water-soluble polymer containing a .beta.-hydroxylalkyleneamine moiety. The Silver method is somewhat disadvantageous in that the polymer must be deposited on the support to ensure enzyme immobilization.
U.S. Pat. No. 4,072,566, issued to Lynn on Feb. 7, 1978, and entitled "Immobilized Biologically Active Proteins", hereinafter referred to as Lynn, discloses a method of bonding enzymes or other biologically active proteins to an inorganic support material using p-phenylenediamine. The support materials disclosed as useful in the invention include siliceous materials, stannic oxide, titania, manganese dioxide, and zirconia. The only supports disclosed in the Examples were 40 to 80 mesh porous glass particles and 120 to 200 mesh porous silica. There is no disclosure in Lynn pertaining to immobilization of proteins upon organic supports such as those made from porous polymers.
U.S. Pat. No. 3,933,589, issued Jan. 20, 1976, to Keyes and entitled "Chemical Immobilization of Enzymes", discloses a method requiring a preformed reaction solution of an alkyl dihalide and an alkane diamine to effect the immobilization.
U.S. Pat. No. 4,251,631, issued Feb. 27, 1981, to Simon, and entitled "Cross-Linked Enzyme Membrane", discloses a method for the preparation of a cross-linked enzyme membrane by directly adsorbing enzymes into the pores of a microporous non-fibrous filter membrane made of a silica modified vinylchloride polymer and then cross-linking the enzyme with a bifunctional coupling agent whereby enzyme molecules are cross-linked to each other without chemically bonding the enzyme molecules to the membrane. The membranes required by the Simon invention are those having finely divided silicon dioxide embedded therein. Further, the cross-linking required by the Simon method requires treatment of the membrane with a cross-linking agent, as for example glutaraldehyde, dioxobenzidine, hexamethylenediisocyanate, or 1,5-difluoro-2,4-dinitrobenzene, after the immobilization of the enzyme on the silica-modified polymer. The enzyme may be immobilized on the silica-modified polymers of Simon prior to treatment of those polymers with a diamine. Immobilization on many other polymers is not possible without such treatment and the Simon process is thus unsuitable for immobilizing enzymes on those other polymers.
U.S. Pat. No. 3,841,969 issued Oct. 15, 1974 to Emery et al and entitled "Preparation of Immobilized Enzymes" discloses a method for preparing a water insoluble enzyme by reacting at a pH of 3 to 7 an enzyme with a titanium, tin, zirconium or iron derivative of a polysaccharide, nylon or glass. For example, a titanic chloride solution may be added to nylon fibers to form a suspension that is dried in an oven overnight. The resulting dry powder is washed three times with an acetate buffer and the buffer and powder separated by centrifugation. The enzyme is added to the resulting slurry and then the water insoluble enzyme formulation is washed with an acetate buffer and sodium chloride solution. Although this procedure is suitable for nylon fibers, it is not suitable for certain other supports that must be treated with a diamine rather than a metal salt before enzymes are successfully immobilized thereon.
U.S. Pat. No. 4,043,869 issued to Barker et al on Aug. 23, 1977 and entitled "Water-insoluble Biologically Active Material" discloses a method of securing enzymes upon water-insoluble solids. Such solids must first be treated with a diazotized N-diaminobenzene and the solids disclosed as suitable supports for the material include inorganics such as porous glass, porous silica and wood.
U.S. Pat. No. 4,204,041 was issued on May 20, 1980 to Bailey et al and is entitled "High Loading of Immobilized Enzymes on Activated Carbon Supports". The method of this invention appears to be suitable only for use with porous particles of activated carbon. The carbodiimides that act as immobilizing agents for the enzymes couple with carboxyl radicals on the surface of the activated carbon. Thus, support materials not having carboxyl radicals on their surface will not be suitably treated by these carbodiimides. Further, as disclosed in column 7 of Bailey, the immobilization is carried out over a period of twenty-four hours.